Recovery of Nontuberculous Mycobacteria and Nocardiae Causing Skin/Soft Tissue Infections Using the Copan ESwab™ Collection and Transport System

Recent reports have shown that infections with nontuberculous mycobacteria (NTM) are increasing worldwide, and often are difficult to diagnose and treat (1,2,3). Consequently, improved diagnostics are needed. The Clinical and Laboratory Standards Institute (CLSI) M40-A2 (4) describes methods for assessing the ability of transport devices to maintain microorganisms in a viable condition for up to 48 hours during transport at room temperature (RT) (20 to 25ºC) and refrigerator temperature (FT) (2 to 7 8ºC). There are no formal published data on the viability of NTM using swab-based systems; however, a study conducted only at RT was presented as a poster at the American Society for Microbiology 110th 9 General Meeting (5). The present investigation was designed using CLSI methods for evaluation of the Copan ESwab™ (Copan Diagnostics Inc., Murrieta, CA) transport system for NTM species commonly causing skin and soft tissue infections.
Nine NTM clinical isolates (Mycobacterium porcinum, M. abscessus subsp. abscessus, M. senegalense, M. mucogenicum, M. fortuitum, M. avium, M. chelonae, M. marinum, and M. haemophilum), previously identified using the Bruker MALDI Biotyper® CA System (Bruker Daltonics Inc., Billerica, MA · USA), and Nocardia asteroides ATCC 19247 were evaluated in this study. After 5 to 7 days growth on blood agar plates (BAP), a heavy stock suspension of each organism was prepared in 4.0 ml sterile saline
19 and vigorously vortexed. From this, suspensions were prepared in triplicate and adjusted to match a 0.5 MacFarland turbidity standard (1.5 ×108 20 CFU/ml) using a Vitek nephelometer (6). Each suspension was serially diluted 1:10 in normal saline to obtain working suspensions of ~ 1.5 ×107 CFU/ml to ~ 1.5×103 22 CFU/ml. One hundred microliters of each working suspension were placed into 18 wells of a microtiter plate. Flocked swabs were placed into the wells and allowed to absorb the inoculum for
approximately 10 seconds and returned into their respective labelled ESwab™ transport vials. Each microorganism/device combination was performed in triplicate for 3 time points (0, 24, and 48 hr). The 0 hr swabs were removed from the transport vials containing 1.0mL Amies broth after ~15 minutes incubation and discarded. 100μL of the inoculum was plated onto a Middlebrook 7H10 agar plate, streaked for isolation, and incubated at 30°C in O2. The remaining ESwabs™ were incubated at RT and FT for 24
and 48 hours. Results were calculated by taking an average of the colony counts from triplicate tests. Counts at 24 and 48 hours were compared to time zero reference counts. Results are summarized in Table 1. The CLSI criterion for acceptable recovery using the roll plate method (i.e., ≥5 colonies recovered from the dilution yielding a baseline count closest to 250 colonies [1]) was met with six of the ten organisms tested at 24hr RT incubation. Of these six, five also met the CLSI criterion at 48hr RT. Another four NTM met the criteria within 10% at 48hr RT. Nine organisms produced CFUs at 24hr and 48hr FT within ~50% of the base line CFUs. The exception was M. marinum, which showed overgrowth after 48 hours of storage. At the 106 39 dilution, seven organisms yielded too numerous to count colonies. At 105 and /or 104 40 dilutions theses same seven yielded countable colonies within ~250 CFU/ml at both 24 and 48 hrs, compared to
zero-hour counts, except M. marinum, which at 105 42 yielded TNTC at 48 hrs RT incubation. M. mu 43 cogenicum, M. fortuitum and N. asteroides had lower counts and produced countable colonies at 10644 . Mycobacterial cells are a bit larger than routine bacteria, thus, initial serial dilutions made from a 0.5McFarland standard contained the desired concentration of bacteria at a higher dilution.
Our results indicate the Copan ESwab™ collection and transport device appears to be an appropriate system for maintenance, transport and recovery of select NTM and Nocardia species by maintaining the viability of NTM known to cause skin and soft tissue infections and an ATCC strain of N. asteroides with fair efficiency for up to 48 hrs at RT. However, until further studies are carried out in the clinical setting to confirm the utility of swab specimens, tissue/fluid samples remain the preferred specimen type for
detection of NTM. Studies directly comparing flocked swabs to paired tissue/fluid specimens are needed. Further study is also required to determine the utility of the ESwab ™ in recovering NTM and Nocardia species from swab specimens collected from patients with suspected infections.